Sperm Head Morphology Alterations Associated with Chromatin Instability and Lack of Protamine Abundance in Frozen-Thawed Sperm of Indonesian Local Bulls

Simple Summary This study analyzed the association of various alterations in sperm morphology with the level of DNA damage, PRM deficiency, and the abundance of the PRM1 gene and protein in sperm. We found in this study that each parameter is associated with one another. Thus, gene expression and PRM1 protein abundance may play an essential role in the stability of sperm chromatin, especially concerning DNA damage and alterations in sperm head morphology. In addition, although it is not a novelty, the CMA3 in this study proved effective for bulls in Indonesia. It can be used as an alternative test to detect PRM content in sperm without having to carry out assessments at the molecular level, such as gene or protein expression assessments. Abstract This study aimed to analyze various alterations in the morphology of the sperm head and its association with nucleus instability and insufficient sperm protamine. Frozen-thawed semen from twenty local Indonesian bulls was used for all stages in this study. The results of sperm head defect assessments are used for bull grouping, high (HD) and low (LD). Sperm DNA damage was assessed using Acridine Orange and Halomax. The PRM1 protein abundance was carried out using an enzyme immunoassay, while PRM1 gene expression was carried out using the RT-qPCR. PRM deficiency was performed using CMA3. Several kinds of sperm head defects in the HD were significantly higher (p < 0.05) than in the LD bulls. Sperm DNA damage showed a significant (p < 0.05) difference between the HD and LD bulls. PRM1 abundance was significantly (p < 0.05) decreased in HD bulls. PRM deficiency was significantly (p < 0.05) higher in HD bulls than in LD bulls. PRM deficiency in bulls correlated significantly (p < 0.01) with sperm head defects, DNA damage, and PRM1 abundance. The lack of sperm protamine might affect the sperm nucleus’s stability and induce morphological alterations in the sperm head.


Introduction
Increasing the local cattle population is the goal of one of the Indonesian government's programs to reduce dependence on imported beef and feeder cattle. The improvement program highlights the various advantages of local cattle, including the excellent adaptability of local cattle to tropical climates, a high level of resistance to various animal diseases, damage, and sperm head defects. In particular, this study presents the latest findings related to these issues in local Indonesian bulls' semen that have not been previously reported.

Experimental Animals and Design
This study used commercial frozen semen and did not involve animals directly. All procedures in semen processing until commercialization follows the operational standards in Singosari AI Center, East Java, Indonesia, namely SNI ISO 9001: 2015 No. G.01-ID0139-VIII-2019 and supervised by a fully responsible veterinarian considering every aspect of animal welfare in Indonesia. The research was conducted in early 2022 during the rainy season in Indonesia. The Singosari AI Center, East Java, provided commercial frozen semen for this study. The frozen semen used was frozen semen from local Indonesian cattle used for the Artificial Insemination program in Indonesia. Twenty Indonesian local bull bulls with an age range of 4 to 5 years, in healthy and productive condition, were used in this study. All bulls were in the same location, with the same type of feed, a body weight of around 700 kg, and an environment with the same average temperature (20-22 • C). Feed was given to the bulls in the form of feed that has been mixed using the TMR (Total Mixed Ratio) method. Complete feed, or TMR, consists of forages and concentrates in balanced and adequate conditions. The feed ingredients included elephant grass, concentrate, silage, hay, and minerals. Fresh semen with more than 70% sperm motility, 800 × 10 6 /mL sperm concentration, and <20% sperm abnormalities was cryopreserved. The frozen semen used in the study had undergone a post-thawing motility check process using computer-assisted sperm analysis (CASA) and met the distribution requirements, progressive motility ≥ 40%.
All bulls used in the study were classified into two groups based on the level of the morphological defects of the sperm head. Evaluation of sperm head morphological defects was carried out using carbol-fuchsin and eosin staining based on Pardede et al. [3]. Two hundred frozen semen straws (ten straws/bull) from twenty local Indonesian bulls were used in this stage. Frozen semen straws were thawed in a water bath at 37 • C for 30 s before being placed in a microtube. The semen samples smeared on glass slides were then fixed with Bunsen and absolute alcohol. After drying, the sample slide was rinsed in a 2% chloramine solution for ±2 min and washed with distilled water and 95% alcohol. Next, the sample slide was stained with carbol-fuchsin and eosin staining for six minutes, washed in running water, and dried. Observations were made with a light microscope with a magnification of 400×. Sperm head morphological defects were counted from a total of 500 sperm cells. Twelve variants of sperm head defects were found and observed in this study, namely abaxial, knobbed acrosome, abnormal contour, macro-micro cephalic, pear shape, undeveloped, round head, detached head, double head, narrow at the base, and tapered ( Figure 1). Sperm head defects from each bull used in the study were calculated as the average. The average value of sperm head defects was 2.96%, ranging from 1.88% to 4.10%. Based on the average value of sperm head defects and the differences from the population average, the bulls were then categorized as high sperm head morphological defects (HD) and low sperm head morphological defects (LD). Our cutoff for driving a wedge into HD and LD categories is the mean of the entire population. Bulls with higherthan-average values of sperm head defects were classified as HD, whereas those with low sperm head defects were classified as LD. Table 1 summarizes the classification of bulls used in this work based on sperm head defects. These grouping results are then used to cluster further assessments, such as DNA damage, PRM deficiency, and PRM1 protein and gene abundance.    Bulls were classified as having high sperm head morphological defects (HD) and low sperm head morphological defects (LD) based on average sperm head morphological defects scores and the percent differences from the population average (p < 0.05).

DNA Damage Assessment by Acridine Orange Staining
Evaluation of DNA damage with acridine orange staining was carried out based on Pardede et al. [12]. Semen from previous sperm head morphological evaluation (200 frozen semen straws (10 straws/bull) from 20 local Indonesian bulls) was used in this stage. The semen samples were first smeared on glass slides and dried in the air. The sample slide was then fixed for two hours in Carnoy's solution (methanol/glacial acetic acid) and air-dried-and, once dry, stained with freshly prepared acridine orange stain for five minutes in the dark. Then, the sample slide was washed with distilled water. Five hundred sperm cells were observed and counted under a fluorescent microscope at 450-490 nm wavelength. Sperm with normal DNA fluoresced green, but sperm with fragmented DNA fluoresced in a yellow-green to orange spectrum ( Figure 2). Bulls were classified as having high sperm head morphological defects (HD) and low sperm head morphological defects (LD) based on average sperm head morphological defects scores and the percent differences from the population average (p < 0.05).

DNA Damage Assessment by Acridine Orange Staining
Evaluation of DNA damage with acridine orange staining was carried out based on Pardede et al. [12]. Semen from previous sperm head morphological evaluation (200 frozen semen straws (10 straws/bull) from 20 local Indonesian bulls) was used in this stage. The semen samples were first smeared on glass slides and dried in the air. The sample slide was then fixed for two hours in Carnoy's solution (methanol/glacial acetic acid) and air-driedand, once dry, stained with freshly prepared acridine orange stain for five minutes in the dark. Then, the sample slide was washed with distilled water. Five hundred sperm cells were observed and counted under a fluorescent microscope at 450-490 nm wavelength. Sperm with normal DNA fluoresced green, but sperm with fragmented DNA fluoresced in a yellow-green to orange spectrum ( Figure 2). The semen samples were first smeared on glass slides and dried in the air. The sample slide was then fixed for two hours in Carnoy's solution (methanol/glacial acetic acid) and air-dried-and, once dry, stained with freshly prepared acridine orange stain for five minutes in the dark. Then, the sample slide was washed with distilled water. Five hundred sperm cells were observed and counted under a fluorescent microscope at 450-490 nm wavelength. Sperm with normal DNA fluoresced green, but sperm with fragmented DNA fluoresced in a yellow-green to orange spectrum ( Figure 2).

DNA Damage Assessment with Sperm-Bos-Halomax
The study of DNA damage with the Sperm-Bos-Halomax was conducted based on Pardede et al. [13]. Another frozen thawed-semen straw was used in this stage. Two hundred frozen semen straws (ten straws/bull) from twenty local Indonesian bulls were used. A total of 50 µL of liquid agarose was put in a tube containing 25 µL of the semen sample (15-20 × 106 cells/mL) at 37 • C and mixed carefully. First, we placed a drop of 1.5-2 µL of cell suspension on the marked wells of the pre-cooled glass plate (4 • C), covered the four wells with a 24 × 24 mm coverslip, and transferred them into the fridge at 4 • C for 5 min. Then, we removed the slides from the refrigerator, carefully removed the coverslip at room temperature (22 • C), and placed them horizontally over the float in a Petri dish. Next, we dried the lysis solution on the slides for 5 min. Next, we washed the slides for 5 min with distilled water and dried them by tilting the slides. Then, the slides were dehydrated in ethanol solution (70% and 90%) for 2 min each and left to dry. In the final step, we stained the slides with 5 µL of 0.01 mm propidium iodide for 5 min. A total of 500 sperm cells were observed under a fluorescence microscope. Sperm with intact DNA has a small halo, whereas sperm with fragmented DNA has a wide halo ( Figure 2).

Measurement of Bovine Sperm Protamine 1 Protein and Gene Abundance
PRM1 protein concentration was measured using enzyme immunoassay (EIA). Another frozen thawed-semen straw was used in this stage. Two hundred frozen semen straws (ten straws/bull) from twenty local Indonesian bulls were used. A 100 µL semen sample was centrifuged for 15 min and washed twice with a phosphate-buffered saline solution (PBS). The precipitated sperm was then used for further EIA. Sperm samples and standard Bovine PRM1 samples were inserted into suitable wells. We covered the reaction wells with adhesive tapes, hatching them in an incubator at 37 • C for 90 min. After that, we washed the plates twice and filled each well with 100 µL of the biotinylated antibody working solution. We covered and hatched them. Then, we returned them to the incubator for 60 min at 37 • C. We rewashed the plate three times, then covered and hatched for 30 min at 37 • C with 100 µL of the enzyme working solution. We then rewashed the plate five times, added 100 µL of the color reagent solution, and hatched for 30 min at 37 • C in a dark incubator. Then, we carefully mixed 100 µL of color reagent C. Using an EIA reader with a wavelength of 450 nm, the concentration of Bovine PRM1 was determined [13].
According to Pardede et al. [13], the PRM1 gene expression level was evaluated using the RT-qPCR technique. At this point, another frozen thawed-semen straw was used. Four hundred frozen semen straws (twenty straws/bull) from twenty local Indonesian bulls were used in this stage. Each frozen semen straw was defrosted in a water bath, washed Animals 2023, 13, 2433 7 of 13 three times with phosphate-buffered saline (PBS), and centrifuged at 16,000× g for 15 min to pellet the sperm (approximately 25 × 10 6 sperm cells/mL). The TRI reagent was then used to extract the total RNA as directed by the manufacturer (Zymo Research, Irvine, CA, USA). The quantity and purity of the total RNA were quantified using the NanoDrop TM One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Marsiling Industrial Estate Rd 3, Singapore). The SensiFAST TM cDNA Synthesis Kit (Bioline Ltd., Memphis, TN, USA) was also utilized for cDNA synthesis, following the manufacturer's guidelines. This procedure produced 20 µL of cDNA that was prepared for RT-qPCR. The quantity of the transcripts was also determined using the quantitative real-time PCR (qPCR), and the reactions were carried out using the SsoFast TM EvaGreen Supermix (Bio-Rad Lab, Hercules, CA, USA). The study's assessed genes included PPIA (XM_001252921.1; forward: ATGCTGGCCCCAACAA-3 and reverse: 5 -CCCTCTTTCACCTTGCCAAA-3 ) as a housekeeping gene and PRM1 (BC108207; forward: AGATACCGATGCTCCTCACC-3 and reverse: 5 -GCAGCACACTCTCCTCCTG-3 ). The PRM1 gene's relative levels of expression were compared to those of the housekeeping gene PPIA using the 2 −∆∆CT method.

Protamine Deficiency Assessment with CMA 3 Staining
The CMA 3 staining method developed by Carreira et al. [11] was used to assess sperm PRM deficiency. Another frozen thawed-semen straw was used in this stage. Two hundred frozen semen straws (ten straws/bull) from twenty local Indonesian bulls were used. The semen samples were smeared on glass slides and fixed in a methanol/glacial acetic acid (3:1) solution for 5 min at 4 • C. Then, for 20 min, we soaked the sample slides in a CMA 3 solution (0.25 mg/mL CMA 3 in McIlvane's buffer (pH 7.0) supplemented with 10 mM MgCl 2 ). The slides were then rinsed in McIlvane's buffer and dried as directed. A fluorescent microscope was used to examine 500 sperm cells. Positive CMA 3 or PRM deficiency in sperm was indicated by bright green fluorescence on the sperm head. In contrast, negative CMA 3 or sperm with intact PRM was indicated by faint green staining on the sperm head.

Statistical Analysis
Twenty bulls (n = 10 HD, n = 10 LD) were utilized for the statistical test. The data with a sperm head defects trait were analyzed using a generalized linear mixed model. Data analysis to determine the grouping of breeding bulls based on the f sperm head defects phenotype has been explained in the previous subsection. The normality test on the research data was carried out using the Shapiro-Wilk test, which was then tested for homogeneity using the Levene test. The research data are normally distributed and vary homogeneously. The statistics were analyzed using SPSS version 25.0 (IBM, Armonk, NY, USA) and the student T-test methodology. In addition, the correlation between CMA 3 and various semen parameters in this study was analyzed using Pearson correlation. The data are presented in tables and graphical diagrams with the mean ± standard error of the mean (SEM).

Correlation of PRM Deficiency, Sperm Head Defects, DNA Damage, and PRM1 Abundance
Sperm PRM deficiency in bulls had a high and significant correlation with sperm head defects (p < 0.000), DNA damage by AO staining (p < 0.000), and the Sperm-Bos-

Discussion
This study analyzed the structure of sperm chromatin to determine the relationship between sperm head morphology alterations with DNA instability and sperm PRM content in local Indonesian bulls. Twelve variants of sperm head defects were observed in this study. These alterations in sperm morphology were significantly positively correlated with DNA damage either by AO staining or by Sperm-Bos-Halomax ® . Abnormal contour, undeveloped, macro-micro cephalic, tapered, narrow at the base, double head, and round head defects are associated with genetic factors, chromosomal abnormalities, DNA damage, and sperm chromatin disorders [14][15][16]. Abnormal chromatin condensation triggers the elasticity of the sperm cell nucleus, resulting in an irregularity in the form of the sperm head that affects the sperm head with an abnormal contour [17]. The undeveloped defect is a defect in sperm in which the sperm does not develop. It can be small, and further examination found that sperm cells with this defect are not composed of complete genetic material [16]. In sperm, microcephalus abnormalities are linked to problems with cell nucleus condensation [18]. In contrast, macrocephalus defects can be caused by genetic factors such as DNA or chromosome abnormalities [17]. The tapering defect in sperm is linked to a breakdown of ectoplasmic specialization in Sertoli and archoplasms cells involved in cell nuclei production [19].
Narrow at the base defects are linked to sperm DNA damage [20]. A disruption in epididymis transit has also been identified as a cause of this condition [21]. Double head defects in sperm occur due to interference with the cytogenesis process or the formation of intracellular bridges during meiotic division, which causes imperfect separation of the sperm [22]. This defect has also been linked to DNA damage [20]. Other sperm head defects reported to have a genetic influence include round-head defects, pear-shaped defects, and knobbed acrosome. A round head defect is a defect in sperm characterized by a round head of the sperm without an acrosome boundary associated with genetic disorders [16]. Pearshaped defects can be caused by abnormal conditions resulting from changes in testicular function, such as testicular temperature regulation or hormonal disturbances [16]. They can be passed on to offspring [16]. Donald and Hancock [23] reported that a high-knobbed acrosome defect is closely related to autosomal recessive sex. A delay in forming the acrosomal phase at the time of spermiogenesis is the cause of this defect [16]. Apart from genetic factors, environmental factors, such as hormonal imbalances, stress, nutritional deficiencies, and extreme climatic conditions, can also influence sperm abnormalities [24].
Overall, each defect in the sperm head in this study did not exceed 1%, although there was a significant difference (p < 0.05) between the HD and LD bulls' groups in several variants of sperm head defects. Chenoweth [16] revealed that bulls with good fertility have at least a proportion of less than 1% in each variation of sperm head abnormalities. Abnormal contour, undeveloped, pear-shaped, macro-micro cephalic, and knobbed acrosome defects in high numbers can cause sperm not to perform its fertilization function and reduce fertility [17]. Detached head and abaxial defects were found in small numbers in this study. The detached head is characterized by separating the head from the sperm tail, which occurs in the epididymis [25]. Chenoweth [16] reported that bulls that were in a sexually inactive status for several weeks experienced a rise in the proportion of detached heads. The abaxial defects found in this study were characterized by the position of the sperm tail, which was not in the middle, and were reported not to affect fertility [16].
However, the various types of alterations in the morphology of the sperm head must be taken into account, mainly if they are observed in large numbers, as they might interfere with male fertility. Bulls with overall morphological defects >17%, according to Ball and Peters [5], do not have good reproductive efficiency. According to Patel et al. [26], the overall morphological defect in bulls was 20%, with general morphological defects ranging from around 8-12 percent in bulls with good fertility. The sperm head defects in this study were classified as usual, namely less than 5%. Pardede et al. [3] reported that sperm with a percentage of total sperm abnormalities of 3-5% have a conception rate of up to 79-84%. The percentage of total sperm abnormalities was >10%; the conception rate only reached <66%. In their study, Petac and Kosec [27] reported that the overall conception rate in bulls with sperm head defects of 4.0% was 73.3%. Another study reported that a bull with 36% sperm head defects resulted in a pregnancy rate of 46% [28]. Anderson et al. [29] also reported that sperm head defects, especially those related to abnormalities in the acrosome area in bovine sperm, with a 23-28% percentage, resulted in an average 60-day nonreturn rate of 48.8-62.3%. Although this study has not analyzed the fertility rate of each bull used, referring to the findings and based on previous studies, perhaps the sperm head defects in this study, which are only less than 5%, can achieve a pretty good percentage conception rate. Even that can be achieved if other factors besides the bull factor in the artificial insemination program can be controlled to achieve optimal fertilization rates.
The stability of sperm DNA in chromatin is one factor that must be maintained, given its essential role in successful fertilization and embryonic growth [3]. This study also found that DNA instability resulted in various alterations in the sperm head. Increased DNA damage was found in bulls in the group with high sperm head abnormality in the bulls. DNA damage assessment was carried out in this study using AO staining and the Sperm-Bos-Halomax ® . Evenson [30] reported that the SCSA (sperm chromatin structure assay) is the most stable test for DNA damage compared to other tests, and AO staining is the most insensitive test compared to the others. Halomax is the most straightforward chromatin DNA test because it is included in a KIT combined with various cell dyes and can be used with light or fluorescence microscopy [30]. However, the sperm chromatin packaging instability will cause DNA damage and disperse, forming a large halo. Both were still usable in this study and significantly correlated with PRM deficiency and sperm head abnormality. DNA damage that occurred in this study as a whole was less than 4%. Previous research reported that DNA damage in sperm that is less than 15% is still expected, whereas 15% to 25% decreases fertility, and sperm with DNA damage more significant than 25% are categorized as sterile [31]. Pardede et al. [31] reported that DNA damage could reach 3-4% in bulls at productive age and still produce a fertility index from in vivo fertilization up to >70%. Thus, DNA damage in this study is still classified as normal, and the bulls have good potential regarding fertility traits. Overall, the formation of sperm morphology, or what is known as the process of sperm morphogenesis, plays an important role in ensuring the normal function of sperm. Various unique and complex mechanisms, including molecular mechanisms, are important keys to the morphological integrity of sperm. The failure of such morphogenesis can lead not only to structural defects of sperm and reduced sperm maturity but also to the fragmentation of sperm nuclear DNA due to impaired repair of damaged nuclear DNA strands during early spermatid stages. Sperm with reduced maturity will produce higher levels of reactive oxygen species and increased DNA damage. Finally, oxidative stress, 'abortive' apoptosis of differentiated germ cells, and teratozoospermia can arise. Furthermore, of the molecular mechanisms involved in the morphogenesis process, one is the mechanism of action of protamine, which is the main protein of the sperm nucleus and is involved in the condensation of spermatid chromatin.
The high PRM abundance in sperm influences DNA stability and the shape of the sperm head [3,8]. In our study, PRM abundance was significantly decreased in the bull group with high head defects. This was followed by significant DNA damage, both by AO staining and the Sperm-Bos-Halomax ® in this study. Moreover, this study demonstrated a close correlation between PRM, DNA instability, and changes in sperm head morphology. Zhu et al. [4] stated that head defects occur due to abnormal spermiogenesis. A sperm chromatin modification process happens at this phase, regulated by histone turnover via PRM and transitional proteins [9]. PRM will replace up to 85 percent of the histone sperm nucleus during this period [8]. PRM, which is the main protein in sperm, has a role in binding DNA, is the primary controller of the presence or absence of DNA damage, and includes changes in sperm head morphology. The disruption of PRM will trigger various alterations, including impaired chromatin compaction that can induce DNA breaks, which can disrupt the DNA backbone and indirectly appears to be the major cause of poor semen morphology. However, more in-depth studies are still needed to enrich the findings in this study, one of which is adding fertility rate data and analyzing its association with various sperm head defects found in this study. In this way, the influence of sperm head defects due to DNA instability and a lack of protamine abundance on the biological aspects of bulls, especially fertility, will be mapped more clearly. CMA 3 staining was significantly inversely associated with high amounts of PRM abundance in sperm in this study. This means that the CMA 3 staining has been proven to be an alternative test for PRM content in sperm. However, this study strengthens and enriches previous studies regarding using CMA 3 to assess chromatin packaging with abnormal or deficient PRM conditions [11]. Furthermore, CMA ' 's fluorochrome-specific guanine-cytosine competes with DNA's PRM binding site. Therefore, chromatin packaging with PRM deficiency conditions will impact the bonding between CMA 3 staining and DNA [11]. In sperm with DNA chromatin packaging, which is compacted and stabilized by PRM, arginine will inhibit CMA 3 staining [32].
Furthermore, the close correlation between the CMA 3 protamine deficiency tester and DNA fragmentation, as well as sperm head defects, further suggests the possibility of CMA 3 being a useful tool that can simultaneously assess all three parameters at once, PRM1 content, DNA fragmentation, and sperm head defects. Unfortunately, this study has not been able to provide fertility data to enrich this study and increase the usefulness of this test. As reported by Nasr-Esfahani et al. [33], CMA 3 staining as an indicator of protamine content, along with semen analysis, could be a useful full test for evaluating fertility status in sub-fertile cases. Although recently fertility assessments have been developed based on sperm genes and protein [34,35], considering that the CMA 3 test has good sensitivity and specificity values and is much easier and cheaper, there is nothing wrong with this test being the right choice for testing bull fertility.

Conclusions
This study proves and strengthens the hypothesis regarding the association between alterations in sperm head morphology and DNA damage, PRM deficiency, and the abun-dance of the PRM1 gene and protein. There were twelve types of sperm head defects found in this study, some of which were higher in the HD bull group. Sperm DNA damage and protamine deficiency were higher in the HD bull group, and conversely, the abundance of the PRM1 gene and protein was lower in the HD bull group. There is a close association between sperm head defects, protamine deficiency, and the abundance of the PRM1 gene and protein. In addition, although it is not a novelty, the CMA 3 in this study proved effective for bulls in Indonesia. It can be used as an alternative test to detect PRM content in sperm without having to carry out tests at the molecular level, such as gene or protein expression tests. Furthermore, considering that CMA 3 is much cheaper than testing at the molecular level, the use of CMA 3 might also be very useful to apply to several developing countries whose economic systems are less stable.
Author Contributions: A.K., B.P.P., M.A. and B.P. designed the study. A.K. and B.P.P. performed all the laboratory work, analyzed the data, and wrote the manuscript. F.A.S., R.I. and Z.N.A.R. performed several laboratory works. A.K., B.P.P., M.A. and B.P. edited and revised the final manuscript. All authors have read and agreed to the published version of the manuscript.